Xylose reductase (XR) catalyzes the first step in xylose metabolism, reducing the pentose sugar to xylitol with the concomitant oxidation of NAD(P)H to NAD(P). This enzyme is important at least in two areas: (1) xylose fermentation for ethanol production and (2) conversion of xylose into xylitol, which is a low calorie food additive. N. crassa was identified as able to convert plant biomass directly into ethanol and is known to possess D-xylose metabolizing enzymes.
Xylose reductase (XR) is an enzyme found commonly in yeast and fungal organisms often with several isozymes in the same species. This enzyme catalyzes the first step in the metabolism of D-xylose and other pentose sugars by reducing the linear aldehyde form of the sugar to xylitol (or a corresponding sugar alcohol). Xylitol can then be oxidized to xylulose by NAD-dependent xylitol dehydrogenase and phosphorylated by D-xylulokinase. The resulting sugar phosphate can enter the pentose phosphate pathway. The reversible reduction of xylitol by XR occurs concomitantly with NAD(P)H oxidation. In general, XR is specific for NADPH, but in some cases it utilizes both NADPH and NADH and in at least one case prefers NADH over NADPH. The different forms of XR in the same species usually have different cofactor preferences and they are likely needed to maintain the redox balance between nicotinamide cofactors under a variety of growth conditions. In order to maintain this balance under anaerobic conditions, XR is likely to be NADH-dependent because the enzyme in the following step (xylitol dehydrogenase) is NAD specific. However, under aerobic conditions either cofactor can be used since cofactors can be regenerated. Some yeast species have solved this problem by utilizing one form of XR with dual cofactor specificity.
Based on sequence and structure similarities, fungal and yeast XRs have been classified as members of the aldo-keto reductase (AKR) enzyme superfamily and more specifically, they belong to the aldose reductase family (EC 1.1.1.21). AKRs have been studied for their ability to detoxify reactive carbonyl compounds, control osmotic pressure by regulating intracellular polyols, and of clinical interest, in diabetic complications resulting from aldose reductase (AR) activity in hyperglycemic patients. The majority of the more than 100 known AKRs are monomeric, however most XRs are homodimers. Other AKRs have quaternary structural organization, but the dimeric interface of XR is unique. Most AKRs favor the reaction in which the carbonyl substrate is reduced. However, their substrate specificity is often very flexible. This is true for XRs as well, which favor production of xylitol and NAD(P) and can often host a variety of other aldehyde substrates.
Although human AR has been studied for decades due to its formation of high levels of polyols in hyperglycemic tissues of diabetic patients, XR in yeast has gained interest for an entirely different reason. D-xylose is known to be among the most abundant sugar constituents of plant biomass as the predominant subunit of hemicelluloses like xylan and xyloglucans. Because XR is critical to xylose utilization by yeast and fingi, this enzyme is important in the fermentation of plant biomass to ethanol. Enhancing the fermentation efficiency is of interest because this fermentation could convert agricultural byproducts and waste into a useful energy source. Improving xylose metabolism may result from recombinant expression of xylose utilizing genes including XR. Additionally, XR may be applied to the production of xylitol, a non-caloric anticariogenic natural sweetener. In this way, XR is linked to human AR because xylitol is a possible sugar substitute for diabetics. Its metabolism is not insulin dependent. An economical means of producing xylitol from xylose in vitro utilizing an XR and a cofactor regeneration system has been proposed by Nidetzky et al. (1996). Similar processes have also been proposed by Ikemi et al. (1990) for converting glucose into sorbitol. Therefore highly active XRs are desirable both for improving xylose metabolism for fermenting yeast and as a reliable low cost source of pure XR for in vitro xylitol production.
Xylitol is usually prepared by processes in which a xylan-containing material is first hydrolysed to produce a mixture of monosaccharides, including xylose. The xylose is then converted to xylitol, generally in a chemical process using a nickel catalyst such as Raney-nickel.
The primary genetic sequences of many XRs have been determined and several have subsequently been cloned and expressed in a variety of hosts. However, a significant lag between genome sequence information and biochemical information has left a large number of proteins, including possible XRs, unidentified. In 2003, the entire 40 Mb genome of the common fungi Neurospora crassa was sequenced. N. crassa has been the subject of over 70 years of research as a model organism for multicellular eukaryotes. A useful characteristic of this organism is that it can directly convert plant biomass to ethanol because it produces cellulase and xylanse enzymes. D-xylose metabolizing enzymes are related to xylose fermentation.